Murine monoclonal antibody protective against Plasmodium vivax malaria

ABSTRACT

The invention relates to a passive protective agent against  P. vivax . The passive protective agent is an antibody that, when a concentration of the antibody is injected intravenously, protects a subject to the limits of that concentration of antibody from developing malaria when the subject is subsequently challenged with live, infectious  P. vivax  sporozoites. The invention includes methods of treatment and pharmaceutical formulations of the agent.

This is a continuation-in-part of application Ser. No. 07/609,549 filedNov. 6, 1990, now abandoned.

SPECIFICATION DEPOSIT INFORMATION

The Hybridoma, NVS3 (Navy Vivax Sporozite 3) is deposited in theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, USA, by a deposit received Nov. 30, 1990,under the terms and conditions of the Budapest treaty for a period ofthirty (30) years. The ATCC designation number is HB 10615, Under theterms of the deposit access to the culture will be available duringpendency of the patent application to one determined by the Commissionerof Patents and Trademarks to those found to be entitled thereto under 37CFR 1.14 and 35 U.S.C. 122, and all restrictions on the availability tothe public of the culture will be irrevocably removed upon grant of thePatent.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a passive protective agent against P. vivax.More particularly this invention relates to an antibody that, when aconcentration of the antibody is injected intravenously, protects asubject to the limits of that concentration of antibody from developingmalaria when the subject is subsequently challenged with live,infectious P. vivax sporozoites.

2. Description of the Prior Art

There have been major efforts toward development of malaria vaccinesundertaken during the past 20 years. Although a commercially viablevaccine has not been achieved to the time this application is filed,there have been successes in providing vaccine protection. The continuedvast investment in vaccine research by both governments world wide andindustry shows an expectation of achieving a commercially viablevaccine. A commercially viable vaccine is one that provides protectionwith minimum side effects, is capable of being produced in quantity, andis stable in storage for a reasonable time under reasonable conditions.These conditions and requirements are well known in the medical andpharmaceutical arts. Even the near misses of total successes (e.g.successes with only a small population) are useful in understanding themechanisms of malaria and further defining the parameters that will leadto a commercially successful vaccine or treatment. The current status ofmalaria vaccine development has been summarized in a recent Institutionof Medicine Report¹. The introduction to the section on vaccines isincluded verbatim to provide part of the background for thisapplication.

WHERE WE ARE TODAY

Prospects for a Vaccine

Vaccination is an exceptionally attractive strategy for preventing andcontrolling malaria. Clinical and experimental data support thefeasibility of developing effective malaria vaccines. For example,experimental vaccination with irradiated sporozoites can protect humansagainst malaria, suggesting that immunization with appropriatesporozoite and liver-stage antigens can prevent infection in individualsbitten by malaria-infected mosquitoes. In addition, repeated naturalinfections with the malaria parasite induce immune responses that canprevent disease and death in infected individuals, and theadministration of serum antibodies obtained from repeatedly infectedadults can control malaria infections in children who have not yetacquired protective immunity. These data suggest that immunization withappropriate blood-stage antigens can drastically reduce the consequencesof malaria infection. Finally, experimental evidence shows thatimmunization with sexual-stage antigens can generate an immune responsethat prevents parasite development in the vector or, offering a strategyfor interrupting malaria transmission.

Prospects for the development of malaria vaccines are enhanced by theavailability of suitable methods for evaluating candidate antigens.These include protocols that allow humans volunteers to be safelyinfected with malaria, and the identification of many areas in the worldwhere more than 75 percent of individuals can be expected to becomeinfected with malaria during a three-month period. In contrast tovaccine for disease of low incidence, for which tens of thousands ofimmunized and non-immunized controls must be studied over several years,malaria vaccines could be evaluated in selected areas in fewer than 200volunteers in less than a year.

Developments in molecular and cellular biology, peptide chemistry, andimmunology provide the technological base for engineering subunitvaccines composed of different parts of the malaria parasite, anapproach that was not possible 10 years ago. During the past 5 years,more than 15 experimental malaria vaccines have undergone preliminarytesting in human volunteers. Although none of these vaccines has provensuitable for clinical implementation, progress has been made in definingthe parameters of a successful vaccine and the stage has been set forfurther advancement.

Despite the inherent attractiveness and promise of this approach, thereremain a number of obstacles to vaccine development. With the exceptionof the erthrocytic (blood) stages of P. falciparum, human malariaparasites cannot be readily cultured in vitro, limiting the ability ofresearchers to study other stages of this parasite and all stages of theother three human malaria parasite species.

In vitro assays, potentially useful for screening candidate vaccines foreffectiveness, do not consistently predict the level of protectiveimmunity seen in vivo. The only laboratory animals that can be infectedwith human malaria parasites are certain species of nonhuman primates,which are not naturally susceptible to these organisms. This makes itdifficult to compare the results of many studies done in animals withwhat happens in human malaria infection.

The promises of modern vaccinology, while potentially revolutionary,have so far proved elusive. Few commercially available vaccines havebeen produced by this technology, for both scientific and economicsreasons. Scientists have not yet been able to assemble defined syntheticpeptides and recombinant proteins and combine them with new adjuvantsand delivery systems into a practical human malaria vaccine. However, asdiscussed above and in the remainder of this chapter, there are goodreasons to believe that this approach will ultimately succeed.

Approaches to Vaccine Development

The complex life cycle of the malaria parasite provides a number ofpotential targets for vaccination. Under investigation are vaccines thatwould be effective against the extracellular sporozoite, during theshort period it spends in the bloodstream; the exoerythrocytic (orliver-stage) parasite, during the roughly seven days it develops withinliver cells; the extracellular merozoite, released from liver cells orinfected erthrocytes and free in the circulation prior to invading othererthrocytes; the asexual parasite that develops within red blood cells;exogenous parasite material released from infected erthrocytes; and thesexual-stage parasite, which occurs both inside erythrocytes and inmosquitoes. The optimal vaccine would include antigens from thesporozoite, asexual, and sexual stages of the parasite, thus providingmultiple levels of control, but vaccines effective against individualstages could also prove highly useful. In addition, a vaccine againstthe Anopheles mosquito itself, which reduced the insect's life span andprevented complete development of the parasite, could be valuable.

Regardless of the stage of parasite targeted for vaccine development, asimilar strategy is envisioned. Based on knowledge of the mechanisms ofprotected immunity, specific parasite antigens (immunogens) areidentified that induce a protective immune response, and synthetic orrecombinant vaccines that accurately mimic the structure of that antigenare prepared.

In the subunit approach to vaccine development, this is done bycombining the immunogen with carrier proteins, adjuvants, and livevectors or other delivery systems. This approach is being pursuedthroughout the world in laboratories studying infectious diseases.Clinical utility has yet to be demonstrated for the majority of theseefforts, and barriers to obtaining satisfactory immunization by thesubunit approach remain. Nevertheless, research on malaria subunitvaccines will continue to be at the cutting edge of this innovative andimportant approach to vaccine development.

It is clear from this description that major advances have been made,and many parasite proteins that could be targets of vaccine developmenthave been identified. What has been lacking is an effective,economically feasible method for inducing protective immune responsesagainst these already identified proteins. Perhaps the most strikingexample has been in the field of pre-erythrocytic stage malaria vaccinedevelopment in which there is already an effective vaccine for humans,the irradiated sporozoite vaccine, but the vaccine is totallyimpractical for widespread human use because of production andadministration problems.

The Irradiated Sporozoite Model

In the 1940s, Mulligen and colleagues² demonstrated that immunization ofchickens with radiation attenuated Plasmodium gallinaceum sporozoiteinduced protective immunity. In the late 1960s, Nussenzweig andcollegues³ demonstrated that immunization of A/J mice with radiationattenuated P. berghei sporozoite protected mice against challenge withinfected erythrocytes were not protected. In the early 1970s Clyde andcolleagues⁴⁻⁶ and Rieckmann and colleagues^(7,8) demonstrated thatimmunization of humans by the bite of irradiated Anopheles speciesmosquitoes carrying P. falciparum and in one case P. vivax sporozoitesin their salivary glands protected these volunteers against challengewith live sporozoites. Like the immunity in mice, this immunity wasstage specific, and it was also species specific; immunization with P.falciparum did not protect against P. vivax . However, it was not strainspecific; immunization with P. falciparum sporozoites from Burmaprotected against challenge with sporozoites from Malaya, Panama and thePhilippines⁴, and immunization with sporozoites from Ethiopia protectedagainst challenge with a strain from Vietnam⁸. These human studies havebeen repeated recently^(9,10) reconfirming that there already is aneffective malaria vaccine, and demonstrating this protective immunitylasts for at least 9 months¹¹. Unfortunately, sporozoites have to bedelivered alive, and since mature, infective sporozoites-infectedmosquitoes, the targets and mechanisms of this protective immuneresponse had to be identified so as to construct a synthetic orrecombinant vaccine.

Of the four human malarias, P. vivax and P. falciparum are the mostcommon and cause the majority of the malaria-induced disease seenworldwide. Prevention of infection by these human parasites wouldalleviate a major health problem in the tropical and subtropical areasof the world. The most promising method for the control of malariaappears to be the development and use of vaccines. One approach tomalaria vaccine development involves the use of the circumsporozoite(CS) protein as a vaccine antigen. This protein covers the surface ofthe sporozoite. The sporozoite is the life stage of the parasite whichis transmitted to humans by feeding female Anopheline mosquitoes.Evidence from both mouse and human malarias indicates that antibodies tothe CS protein can provide protection in vivo against infection bysporozoites (Charoenvit et al., Infect. Immunity 55:604, 1987;Charoenvit et al., J. Immunol., 146, pp. 1020-1025, (1991). Khusmith etal., Science, 252, pp. 715-718, (1991).

In 1985, McCutchan and colleagues sequenced the gene for the CS proteinin P. vivax and determined the amino acid sequence derived from thatgene, (McCutchan et al., Science 230:1381, 1985). The monoclonalantibody originally used by McCutchan and colleagues (McCutchan et al.,Science 230:1381, 1985) to identify the protein and isolate thenucleotide sequence which later became the subject of theMcCutchan/Wistar U.S. Pat. No. 4,693,994 was originally developed byCharoenvit and Beaudoin of the Infectious Diseases Department, NavalMedical Research Institute (NMRI). This monoclonal antibody is themonoclonal antibody of this invention. It has been named or designatedMAB Navy Vivax Sporozoite 3 (NVS3). In 1987, McCutchan and Wistar, inU.S. Pat. No. 4,693,994, described a repeated nine amino acid sequencewithin the CS protein as an immunodominant synthetic peptide. Therepeated sequence is Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala.

In the '994 patent and in other publications, McCutchan/Wistar maintainthat the nine amino acid sequence is capable of inducing antibodiesprotective against P. vivax malaria. Experimental evidence indicatesthat while the McCutchan/Wistar sequence stimulates the development ofanti-CS antibody in humans, it has not been shown to induce protectiveantibodies. In an article published in Am. J. Trop. Med. Hyg. 40(5),p455-464 (1989), Collins et al. describes tests in which Saimiri monkeys(Saimiri sciureus boliviensis), which are susceptible to human vivaxmalaria, were immunized with two different preparations (VIVAX-1 andNS1₈₁V20). Both preparations contain the McCutchan/Wistar peptide(Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). When these monkeys werechallenged with 10⁴ P. vivax sporozoites, there was no significantprotection.

Nussenzweig et al., in U.S. Pat. No. 4,826,957, describes an immunogenicrecombinant yeast expression product which contains a long sequenceincorporating a portion of the P. vivax circumsporozite. The Nussenzweiget al. sequence contains multiple repeats of the sequenceGly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala as part of a complex polypeptide.When used as a vaccine, this polypeptide causes the formation ofantibodies, the antibodies are directed atGly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala and did not provide significantprotection against challenge with sporozoites.

In U.S. Pat. No. 4,957,869, Arnot et al. describes an immunogenicpeptide corresponding to P. vivax CS protein consisting of at least tworepeats of the amino acid sequence Asp-Arg-Ala-X-Gly-Gln-Pro-Ala-Gly. Xis defined as selected from the group consisting of Asp and Ala. Theprior art approachs the problem from the premise that a vaccine isneeded to provide protection against malaria. There is also a need for asimple material to protect against P. vivax.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is a monoclonal antibody whichprovides passive protection against P. vivax.

Another object of the invention is a pharmaceutical preparation whichprovides passive protection against P. vivax.

An additional object of this invention is a means of providing temporaryor limited protection against P. vivax by binding a particular site onthe CS protein of P. vivax and thereby preventing infection bysporozoites of that parasite.

A further object of this invention is an agent to produce and isolate ahuman protective antibody against P. vivax.

Yet an additional object of this invention is a method of using theunique binding and protective nature of the mouse monclonal antibody asa special reagent for conversion into a human monoclonal antibody whichretains the same binding specificity and can therefore be used in humansto induce temporary antibody-mediated passive immunity.

Other objects and advantages of this invention will become clear as thedetailed description of the present invention is presented. These andadditional objects of the invention are accomplished by a murine, IgG3monoclonal antibody designated NVS3 produced by immunizing mice withirradiated P. vivax sporozoites and pharmaceutical preparations of NVS3which neutralize infectious sporozoites of P. vivax.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtainedby reference to the following Description of the Preferred Embodimentsand the accompaning drawings in which like numerals in different figuresrepresent the same structures or elements. The representations in eachof the figures is diagrammatic and no attempt is made to indicate actualscales or precise ratios. Proportional relationships are shown asapproximations.

FIG. 1 is a graph plotting antibody-octapeptide reactivity of peptidescontaining the entire AGDR sequence (AGDR+) and those with part or none(AGDR−) against percent of the optical density of the positive control.

FIG. 2 is a bar chart in which antibody-peptide binding is expressed asthe percent of the positive control optical density (OD of anti-PLAQmonoclonal antibody with PLAQ).

FIG. 3 is a graph of (AGDR)₂ and the recombinant vaccine VIVAX-1incubated with aliquots of sera from monkeys immunized with NS1₈₁V2O.Final serum concentration was 1:250, final peptide concentrations aredepicted along the X-axis. Vertical bars depict standard error.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention rests on the development of a monoclonal, antibodydesignated NVS3 which binds to a particular epitope on the CS protein ofthe sporozoite of the human malaria parasite P. vivax. NVS3 (Navy vivaxsporozoite 3) is an IgG3 isotype antibody. It is species and stagespecific; it reacts only with sporozoites of P. vivax, and does notreact with sporozoites of P. falciparum, P. berghei, P. yoelii, or P.gallinaceum. It is also nonreactive with blood stages of P. vivax, P.falciparum, P. berghei, P. yoelii and P. gallinaceum when tested in animmunofluorescent antibody technique (IFAT). NVS3 is not strainspecific. It reacts with sporozoites from other strains of P. vivax(i.e. North Korean, Sal 1, Colombian, and Thai strains). Western blotanalysis of a P. vivax sporozoite extract showed that NVS3 reacted withfour antigen bands with relative molecular weights of 46, 49, 50 and 57kda. The monoclonal antibody-producing cell, Hybridoma, NVS3 (Navy VivaxSporozoite 3), was submitted for deposit Nov. 30, 1990, under theprovisions of the Budapest Treaty, with the American type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209,USA. Its deposit number is HB10615. The MAB is available from the ATCCwhen this application issues as a patent or upon request to the NavelMedical Research Institute, Bethesda Md. 20889-5055. NVS3 is producedthrough known techniques by immunization with irradiated, but otherwiseintact sporozoite, and not by a recombinant protein.

The unique specificity of this antibody permits pharmaceuticalformulations of the antibody to be administered to a host subject wherethe antibody binds to P. vivax sporozoites in the circulation of thehost and renders the sporozoites noninfectious thereby preventingmalaria disease. The adjuvants and diluents are the pharmaceuticalmaterials usually used for this type of protein material. The dosagewill vary with the subject receiving it.

The unique aspect of this NVS3 antibody lies in the conformation of theantigen binding site (complementarity determining regions orhypervariable regions) of the heavy and light chains of the IgGmolecule. Current technology permits the conversion of this mouse IgGmolecule into a human IgG molecule which still retains the same antigenbinding specificity. Conversion allows the use of the NVS3 antibody as apassive immunization agent similar to the hyperimmune gamma-globulinused to passively immunize against hepatitis A.

The production of a mouse-human “chimeric” or a “humanized” mouse Mabrequires as a starting point a biologically active (in this case“protective”) variable or hypervariable region of a mouse Mab. It shouldbe noted that in the case of Mabs against circumsporoziote proteins, itis only this region that is required since Fab fragments provideprotection in passive transfer. There are a number of publishedstrategies that are employed to accomplish this humanization. The workis routine but tedious. Using NVS3 cell line one would extract RNA anduse known primers to produce heavy and light chain variable regioncDNAs. These would be sequenced using standard methods or machines. Theheavy and light chain CDR sequences are predicted using establishedmethods and alignment with other known heavy and light chain CDRsequences. Having established the sequence of the framework region ofthe variable region, one scans databases to identify sequences of humanIgG with homology to the variable framework region of NVS3 . One wouldthen synthesize heavy and light chains that include the NVS3 CDRsequences and the homologous human IgG framework.

Epitope mapping studies demonstrated that NVS3 recognizes only four(Ala-Gly-Asp-Arg (AGDR)) of the nine amino acids (DRA A/D GQPAG) withinthe repeat region of the P. vivax circumsporozoite protein. Sera frommonkeys immunized with a recombinant protein did not produce antibodiesto this protective epitope. They did, however, produce high levels ofantibodies to other epitopes in the repeat region. The data clearlydemonstrate that circulating antibodies to a defined epitope on the P.vivax CS protein can protect against malaria in vivo, and indicate thatdetermination of the fine specificity of protective antibodies and theconstruction of subunit vaccines to exclude irrelevant amino acidresidues may be critical to the induction of antibodies having theappropriate specificity for mediating protective immunity. The inventionpertaining to the AGDR sequence is the subject of a concurrently filedapplication number 609,551 filed in the names of Hoffman, Charoenvit,and Jones and titled PROTECTIVE FOUR AMINO ACID EPITOPE AGAINSTPLASMODIUM VIVAX MALARIA now U.S. Pat. No. 5,095,093 , issued Mar. 10,1992.

It is noted that those technical terms or phrases used here which havenot been specifically defined have the same meaning as generallyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Having described the invention, the following examples are given toillustrate specific applications of the invention including the bestmode now known to perform the invention. These specific examples are notintended to limit the scope of the invention described in thisapplication.

EXAMPLE 1 Materials and Methods

Animals

Female, 6-8 week old, BALB/c Byj mice (Jackson Laboratories, Bar Harbor,Me.) were used in the production of monoclonal antibodies. Saimirisciureus boliviensis monkeys were used in the passive transfer study toevaluate the protective efficacy of a selected monoclonal antibody. Themonkeys were of Bolivian origin. All animals were quarantined for aone-month conditioning period, weighed, tested for tuberculosis andexamined for concurrent intestinal and blood stage infections.

Sporozoites

P. vivax sporozoites of the Vietnam strain (ONG/CDC), North Korean (NK)and Colombian strains were used for production and characterization ofthe monoclonal antibodies. Sporozoites were separated from infectedAnopheles stephensi mosquitoes by a discontinuous gradient techniquedescribed by Pacheco, N. D., C. P. A. Strome, F. Mitchell, M. P. Bawdenand R. L. Beaudoin; Rapid, large-scale isolation of Plasmodium bergheisporozoites from infected mosquitoes; J. Parasitol; 65:414-417; 1979.Sporozoites of the Salvador I strain were reared in A. stephensimosquitoes by membrane feeding the mosquitoes on blood from agametocytemic chimpanzee as described by Collins, W. E., H. M. McClure,R. B. Swenson, P. C. Mehaffey and J. C. Skinner; Infection of mosquitoeswith Plasmodium vivax from chimpanzees using membrane feeding. Am. JTrop. Med. Hyg.; 35:56-60; 1986. Sixteen days post-feeding, thesporozoites were dissected from the salivary glands of the infectedmosquitoes for use in the challenge studies.

EXAMPLE 2

Production and Characterization of Monoclonal Antibodies

Mice were immunized intravenously at weekly intervals with 3-5×10⁴,radiation attenuated (10⁴ rads) sporozoites. Three days after the thirdimmunization, spleen cells were isolated and fused with X63.Ag8.653,non-immunoglobulin secretor mouse myeloma cells using the well knownmethod described by Kohler and Milstein (75) with a slight modification.Briefly, spleen cells isolated from immunized mice were fused withmyeloma cells using 30% polyethylene glycol (approximate mol. wt. 1000)as a fusing agent. The cells were washed, resuspended in HAT selectivemedium and plated into 96-well tissue culture plates and allowed to growat 37° C. in 5% CO₂ in air. Three weeks later the supernatants from thegrowth wells were screened for antibodies to P. vivax sporozoites usingan immunofluorescent antibody technique (IFAT) as described byCharoenvit, Y., M. F. Leef, L. F. Yuan, M. Sedegah and R. L. Beaudoin;Characterization of Plasmodium yoelii monoclonal antibodies directedagainst stage-specific sporozoite antigens; Infect. Immunol.;55:604-608; 1987. The positive hybrids were cloned by limiting dilution,and the supernatants from the wells containing hybridoma clones wereretested. IFAT positive clones were expanded for the production ofascitic fluid; monoclonal antibodies of interest were purified fromascitic fluid. Double immunodiffusion against goat anti-mouseimmunoglobulins was used to determine isotype. Species and stagespecificities were determined in an immunofluorescent antibody techniqueagainst sporozoites and blood stage parasites from P. vivax, P.falciparum, P. berghei, P. yoelii and P. gallinaceum. Reactivity todifferent strains of P. vivax (North Korean, Salvador 1, Colombian andThai) was also measured. Western blot analysis was used to determine thenumber of proteins in the sporozoite to which the monoclonal antibodyselected for passive transfer binds. The selected antibody wasdesignated NVS3 (Navy Vivax Sporozoite 3). It was purified bystaphylococcal protein A column as described by Hjelm, H. and J.Sjoquist; The use of matrix-bound protein A from Staphylococcus aureusfor the isloation and determination of immunoglobulins; In:Immunoadsorbents in Protein Purification; E. Ruoslahti, editor.University Park Press, Inc. Baltimore; pp.51-57; 1976. The cell lineproducing the NVS3 is deposited with the American Type CultureCollection. The accession number is HB10615.

EXAMPLE 3

Sporozoite Challenge Study

An initial set of experiments was performed to determine the amount ofintravenously injected NVS3 required to achieve antibody levels whichgave responses in an enzyme-linked immunosorbant assay (ELISA) similarto sera from monkeys immunized with NS1₈₁V20 reported by Collins, W. E.,R. S. Nussenzweig, W. R. Ballou, T. K. Ruebush II, E. H. Nardin, J. D.Chulay, W. R. Majarian, J. F. Young, G. F. Wasserman, I. Bathurst, H. L.Gibson, P. J. Barr, S. L. Hoffman, S. S. Wasserman, J. R. Broderson, J.C. Skinner, P. M. Procell, V. K. Filipski and C. L. Wilson; Immunizationof Saimiri sciureus boliviensis with recombinant vaccines based on thecircumsporozoite protein of Plasmodium vivax; Am. J Trop. Med. Hyg.;40:455-464; 1989. Based on these experiments, 2 mg of NVS3 per monkeywas selected for injection intravenously into six Saimiri monkeys. Ofcourse the dosage can vary from 2 to 30 mg dependant on weight andmetabolism of the subject.

An IgG3 monoclonal antibody directed against Trypanosoma bruceirhodesiense in accordance with the method described by Hall, T. and K.Esser; Topologic mapping of protective and nonprotective epitopes on thevariant surface glycoprotein of the WRATat 1 clone of Trypanosoma bruceirhodesiense; J. Immunol.; 132:2059-2063; 1984 was inoculated intoanother six monkeys to serve as an unrelated antibody control group.Nine other monkeys served as uninjected controls. One hour afterantibody transfer, 10⁴ P. vivax sporozoites diluted in normal salinecontaining 10% normal Saimiri monkey serum were injected into allmonkeys. Serum samples were collected prior to antibody inoculation andone hour later (immediately before sporozoite challenge). All animalswere splenectomized 6 to 7 days after sporozoite inoculation. Beginning14 days after sporozoite inoculation and continuing through day 56,giemsa-stained thick and thin blood films were prepared daily.Parasitemias were quantified and recorded per mm³ of blood.

Four of the six monkeys inoculated with NVS3 were fully protectedagainst blood stage disease. The remaining two developed patentparasitemias after 31 and 40 days (Table 1). Five of six monkeysinoculated with the unrelated monoclonal antibody (anti-trypanosoma)developed detectable parasitemias within IS to 24 days (mean=20.6 days)while the nine control monkeys all developed detectable parasitemias in17 to 30 days (mean=20.1 days). The two unprotected monkeys thatreceived NVS3 had longer prepatent periods than the control monkeyswhich received the anti-trypanosoma antibody (p<0.01) and longer thanthe uninjected controls (p<0.005). At splenectomy, the spleens wereobserved to be enlarged in animals of both groups that received themonoclonal antibodies. None of the nine untreated control animals had anenlarged spleen.

EXAMPLE 4

Epitope scanning

A hypothetical peptide containing the repeat regions of the CS proteinsof 4 strains of P. vivax was designed. The four strains were Belem(Arnot, D. E., J. W. Barnwell, J. P. Tam, V. Nussenzweig, R. S.Nussenzweig and V. Enea; Circumsporozoite protein of Plasmodium vivaxgene cloning and characterization of the immunodominant epitope;Science; 230:815-818; 1985), Sal 1 (McCutchan, T. F., A. A. Lal, V. F.de la Cruz, L. H. Miller, W. L. Maloy, Y. Charoenvit, R. L. Beaudoin, P.Guerry, R. Wistar, Jr., S. L. Hoffman, W. T. Hockmeyer, W. E. Collinsand D. Wirth; Sequence of the immunodominant epitope for the surfaceprotein on sporozoites of Plasmodium vivax; Science; 230:1381-1383;1985), North Korean (Arnot, D. E., J. W. Barnwell and M. J. Stewart;Does biased gene conversion influence polymorphism in thecircumsporozoite protein-encoding gene of Plasmodium vivax; Proc. Natl.Acad. Sci. USA; 85:8102-8106; 1988) and VS 210 (Rosenberg, R., R. A.Wirtz, D. E. Lanar, J. Sattabongkot, Ti Hall, A. P. Waters and C.Prasittisuk; Circumsporozoite protein heterogeneity in the human malariaparasite Plasmodium vivax; Science; 245:973-976; 1989). The sequence ofthe peptide is as follows: GDRADGQPAGDRADGQPAGDRADGQAAGNGAGGQPAGDRAAGQPAGDGAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRADGQAAGNGAGGQAAGNGAGGQPAGDRAAGQPAGDRAAGQPAGDRAAGQAAGNGAGGQAA. The methods of Geysen and colleaguesdescribed in Use of peptide synthesis to probe viral antigens forepitopes to a resolution of a single amino acid; Proc. Natl. Acad. Sci.USA; 81:3998-4002; 1984; Small peptides induce antibodies with asequence and structural requirement for binding antigen comparable toantibodies raised against the native protein; Immunol; 82:178-182; 1985;A priori delineation of a peptide which mimics a discontinuous antigenicdeterminant; Molec. Immunol; 23:709-715; 1986; Strategies for epitopeanalysis using peptide synthesis; J. Immunol. Methods; 102:259-274; 1987were employed to synthesize 137 sequential octapeptide subsets of this144 amino acid peptide. The octapeptides were synthesized on the tips ofpolypropylene pins set in 96 pin blocks (Cambridge ResearchBiochemicals, Valley Stream, N.Y.). Octapeptide n=amino acid n throughamino acid n+7. The syntheses were carried out in the wells of 96 wellplates thereby allowing each pin to hold a different amino acidsequence. Conventional Fmoc solid phase methods were used to completethe syntheses. The tetrapeptides PLAQ (and monoclonal antibody to it)and GLAQ were used as positive and negative controls in each set of 96pins. The ability of the monoclonal antibody NVS3 to bind to thepeptides was tested in an ELISA. Each pin was incubated overnight at 4°C. in NVS3 at 2 μg antibody/ml. After washing, the pins were incubatedfor one hour at 37° C. in goat anti-mouse IgG (Kirkegaard and Perry,Gaithersburg, Md.) at a dilution of 1:2000. Optical densities weremeasured after the pins were incubated in substrate (ATBS,2,2′-azino-di-[3-ethyl-benzthiazoline sulfonate] and hydrogen peroxide)for 30 minutes.

Analysis of the ELISA results revealed a correlation betweenNVS3-octapeptide binding and the presence of the tetrapeptide AGDR(alanine-glycine-aspartic acid-arginine) (FIG. 1 in whichantibody-octapeptide reactivity of peptides containing the entire. AGDRsequence (AGDR+) and those with part or none (AGDR−) is plotted againstpercent of the optical density of the positive control. The n values arethe total number of octapeptides containing AGDR (50) and not containingAGDR (87). The positive control optical density was obtained with ananti-PLAQ monoclonal antibody.).

Octapeptides not containing the sequence AGDR were not bound by NVS3.Octapeptides containing subsets of AGDR (e.g. AGD and GDR) were also notreactive. No correlation between reactivity and the location oftetrapeptide within the octapeptide was noted (FIG. 2 in whichantibody-peptide binding is expressed as the percent of the positivecontrol optical density (OD of anti-PLAQ monoclonal antibody with PLAQ).Bars one through eight represent the mean binding of peptides having thesequences shown in the inset. The number above each bar is the n of thatgroup.).

EXAMPLE 5

The following materials are needed for tests.

Peptide Synthesis

The 8-residue peptide (AGDR)₂ was synthesized by the stepwisesolid-phase method of Merrifield, R. B; Solid phase peptide synthesis.I. The synthesis of a tetrapeptide; J. Am. Chem. Soc.; 85:2149-2154;1963. Pam-t-Boc-L-arginine (Tos) resin (0.5 used as the starting pointof the synthesis. The protected peptide resin was deprotected byhydrogen fluoride/p-cresol (9:1, v/v for 1 hour at 0° C.).

Recombinant P. vivax Protein

VIVAX-1 is a recombinant protein containing approximately 60% of theentire CS protein from the Belem strain of P. vivax. It contains therepeat regions (DRA A/D GQPAG)₂₀ (Barr, P. J., H. L. Gibson, V. Enea, D.E. Arnot, M. R. Hollingdale and V. Nussenzweig; Expression in yeast of aPlasmodium vivax antigen of potential use in a human malaria vaccine; J.Exp. Med.; 165:1160-1171; 1987). NS1₈₁V20 (SKF/WRMAIR) vaccine is afusion protein from Eschertichia coli that contains the 20 copies of thenonapeptide repeat present in the repeat region of the CS protein and 81amino acids derived from the nonstructural protein gene of influenza A(Collins, W. E., R. S. Nussenzweig, W. R. Ballou, T. K. Ruebush II, E.H. Nardin, J. D. Chulay, W. R. Majarian, J. F. Young, G. F. Wasserman,I. Bathurst, H. L. Gibson, P. J. Barr, S. L. Hoffman, S. S. Wasserman,J. R. Broderson, J. C. Skinner, P. M. Procell, V. K. Filipski and C. L.Wilson; Immunization of Saimiri sciureus boliviensis with recombinantvaccines based on the circumsporozoite protein of Plasmodium vivax; Am.J Trop. Med. Hyg.; 40:455-464; 1989).

EXAMPLE 6

Immunofluorescent Antibody Technique

NVS3 activity was measured in the serum of the monkeys which receivedintravenous NVS3 prior to sporozoite challenge. Two-fold serialdilutions of sera were used in an IFAT with P. vivax sporozoites as thetarget antigen (Charoenvit, Y., M. F. Leef, L. F. Yuan, M. Sedegah andR. L. Beaudoin; Characterization of Plasmodium yoelii monoclonalantibodies directed against stage-specific sporozoite antigens; Infect.Immunol.; 55:604-608; 1987). To determine if NVS3 reacts with epitopesother than AGDR on sporozoites, aliquots of NVS3 at a concentration of2.5 μg/ml were preincubated with varying amounts of the P. vivax peptide(AGDR)₂ or the unrelated peptide (QGPGAP)₂, a peptide from the repeatregion of P. yoelii CS protein. The antibody-peptide mixtures were thenincubated with P. vivax sporozoites and evaluated by IFAT to determinethe ability of (AGDR)₂ to block the binding of NVS3 to sporozoites.

The immunofluorescent antibody studies show that NVS3 binds to P. vivaxsporozoites but not to P. yoelli sporozoites. Furthermore, this bindingis to a specific epitope; NVS3 binding to P. vivax sporozoites can beblocked by preincubation with the P. vivax octapeptide (AGDR)₂ but notwith the P. yoelii dodecapeptide (QGPGAP)₂.

EXAMPLE 7

ELISA

NVS3 concentrations in sera from monkeys receiving NVS3 prior tosporozoite challenge was measured in ELISA using (AGDR)₂ as the targetantigen. Serum dilutions (1:100) were incubated with (AGDR)₂-coatedwells. The secondary antibody was horseradish peroxidase-labelled goatanti-mouse IgG. Optical density values for the serum samples werecompared with standard values obtained by measuring the reactivity to(AGDR)₂ of known concentrations of NVS3 diluted in equivalentconcentrations of Saimiri monkey serum.

Serum Levels of NVS3 and Anti-(AGDR)₂, Activity

Serum samples from the monkeys passively immunized with NVS3 wereassayed for anti-sporozoite and anti-(AGDR)₂ activities. NVS3 passivetransfer sera contains high levels of antibodies as determined by IFATand ELISA (Table 2).

EXAMPLE 8

Inhibition of Antibody Activity in Sera From NS181V20-immunized Monkeys

Serum from six monkeys immunized with NS1₈₁V20 were tested in ELISA foractivity to (AGDR)₂ and VIVAX-1. Aliquots of each serum sample (1:250final concentration) were incubated with varying concentrations of(AGDR)₂ or VIVAX-1 to determine if activity to the repeat region of theCS protein can be blocked. VIVAX-1 was used as the target antigen in anELISA and P. vivax sporozoites were used as the target antigen in aparallel series of IFAT assays. In both cases, secondary antibody wasgoat anti-human IgG.

Antibody Activity in Sera From Monkeys Immunized with NS1₈₁ V20

Serum samples (1:100 and 1:500 final concentrations) from monkeysimmunized with NS1₈₁V20 reacted will with VIVAX-1 but not with (AGDR)₂in a direct ELISA. When these sera were preincubated with VIVAX-1, allanti-VIVAX-1 activity was removed; preincubation with (AGDR)₂ removed noactivity (FIG. 3 wherein (AGDR)₂ and the recombinant vaccine VIVAX-1were incubated with aliquots of sera from monkeys immunized withNS1₈₁V2O. Final serum concentration was 1:250, final peptideconcentrations are depicted along the X-axis. Vertical bars depictstandard error.). When similarly preincubated serum samples were testedin IFAT for anti-sporozoite activity, VIVAX-1 preincubation eliminatedall anti-sporozoite activity in a VIVAX-1 concentration-dependentmanner. Preincubation with (AGDR)₂ removed no activity (data not shown).

EXAMPLE 9

Inhibition of Liver Stage Development

The ability of NVS3 to inhibit the in vitro development of sporozoitesin hepatocytes was measured following the technique of Millet andcolleagues (Millet, P., W. E. Collins, L. Herman and A. H. Cochrane;Plasmodium vivax: In vitro development of exoerythrocytic stages insquirrel monkey hepatocytes and inhibition by an anti-P. cynomolgimonoclonal antibody; Exp. Parasitol; 69:91-93; 1989). Briefly, a monkeyliver fragment was dissociated by collagenase perfusion and plated in 35mm petri dishes. Equal volumes of serum (or NVS3) and sporozoitesuspension were mixed and incubated at room temperature for 15 minutes.The NVS3-sporozoite mixtures were exposed to the hepatocytes for 2 hoursthen washed. Seven days post-exposure, the monolayers were fixed andschizonts counted microscopically.

Inhibition of Liver Stage Development

After incubation with either serum or NVS3, 2.5×10⁴ P. vivax sporozoiteswere added to each monolayer of primary cultures of Saimiri hepatocytes.Results (Table 2) are expressed as the number of schizonts in twomonolayers. Serum from NVS3-treated monkeys was very effective inreducing or eliminating schizont development.

The studies described here demonstrate for the first time thatcirculating antibodies to human malaria sporozoites can protect againstsporozoite challenge. Four of six monkeys were completely protected; theremaining two had significantly prolonged prepatent periods whencompared to control animals. Circulating antibodies to the P. vivax CSprotein can protect against sporozoite-induced malaria. Although therehas been a major emphasis on protection against malaria by activeimmunization with subunit vaccines, the above examples and data suggestthat another strategy for protecting humans against malaria may be touse human monoclonal antibodies with specificities similar to NVS3 toprovide passive protection during-short term exposure.

EXAMPLE 10

The monoclonal antibody NVS3 is used as a passive prophylactic agent bysolubilization in an appropriate pharmaceutical injectable such as butnot restricted to normal saline and subsequent injection into personsneeding prophylactic protection from P. vivax malaria. The dose of theinjected antibody will be adjusted to provide a protective level ofcirculating antibody. A dose between 50 to 1000 mg per individual may bepreferred. The route of injection may be intravenous, intramuscular orsubcutaneous.

EXAMPLE 11

The monoclonal antibody produced by the above cited hybridoma cell lineis to be humanized by a method which will replace all of the mouseantbody molecule, except the antigen binding site with human antibody.This is genetically engineered by the method of Morrison et al. (PNASUSA, 81:6851, 1984), or other appropriate methods. mRNA from theNVS3-producing hybridoma is isolated and RNA-dependent DNA polymerase isused to produce an RNA/DNA hybrid. The RNA is then removed by treatmentwith RNAase. A Klenow fragment of DNA polymerase 1 is used to makedouble stranded DNA and treatment with EcoR 1 methylase blocks any EcoR1 sites within the strand. EcoR 1 linkers are ligated to the ends of thecDNA which is then treated with EcoR 1. Selective ethanol precipitationis used to separate the cDNA from the linkers. The cDNA is then ligatedinto the EcoR 1 site of a lambda phage (e.g. g+11) and DNA is packagedin a commercial packaging extract. Once E. coli are exposed to thephages, they are plated and screened with cloned V_(H) and V_(K) or 1genes as probes. The V_(H) gene is spliced to human IgG C region geneusing Sal 1 linkers. The V_(K or 1) gene is spliced to the human K or 1light chain joining and C region exons. Both these chimeric geneconstructs are then inserted into a vector. Both vectors must have theability to grow in E. coli, possess a mammalian promoter and havedifferent mammalian and bacterial drug resistance genes. The V_(H) andV_(K) or 1 containing constructs are transfected sequentially into anappropriate mouse myeloma cell line, such as J558L, by calcium phosphateprecipitation. After one construct has been transfected into the hostcell line, successful transfectants are selected for using the drugresistance gene in the vector. Successfully transfected cells thenreceive the second construct and are selected for by use of the second,and different, drug. The cells are then cloned by limiting dilution.Production of antibody having specificity for AGDR in ELISA will be usedto screen the clones.

EXAMPLE 12

The humanized monoclonal antibody possissing the complimentaritydetermining region of NVS3 is used as a passive prophylactic agent bysolubilization in an appropriate pharmaceutical injectable such as butnot restricted to normal saline and subsequent injection into personsneeding prophylactic protection from P. vivax malaria. The dose of theinjected antibody will be adjusted to provide a protective level ofcirculating antibody. A dose between 50 to 1000 mg per individual may bepreferred. The route of injection may be intravenous, intramuscular orsubcutaneous.

TABLE 1 Prepatent periods and maximum parasitemia in monkeys passivelyimmunized with the monoclonal antibody NVS3 and challenged with 10⁴sporozoites of the Salvador I strain of Plasmodium vivax. MaximumMaximum Monkey Prepatent Parasitemia Parasitemia No. Antibody Period permm³ Day SI-74  NVS3/2 mg 40  9,393 56 SI-162 NVS3/2 mg NI — — SI-218NV53/2 mg 31 21,018 55 SI-250 NVS3/2 mg NI — — SI-251 NVS3/2 mg NI — —SI-312 NVS3/2 mg NI — — SI-323 Try/2 mg NI — — SI-319 Try/2 mg 23   34131 SI-330 Try/2 mg 15 42,966 51 SI-316 Try/2 mg 24 47,616 46 SI-321Try/2 mg 23 55,614 39 SI-328 Try/2 mg 18 98,394 56  SI-311* None 29   21 37  SI-238* None 18 30,132 42 SI-320 None 17 30,876 53 SI-45* None30 39,432 56 SI-249 None 19 48,918 56 SI-174 None 20 75,888 45 SI-289None z1 77,748 54  SI-101* None 17 93,588 43 SI-300 None 19 312,000  55*Animals splenectomized on day 6; all others on day 7.

TABLE 2 NVS3 serum levels one hour after passive transfer as measured byimmunofluorescent antibody technique with Plasmodium vivax sporozoitesas antigen, ELISA with (AGDR)₂ as antigen and inhibition of sporozoitedevelopment in Saimiri monkey hepatocytes. Monkeys SI-74 through SI-312received 2 mg of NVS3 while monkeys SI-316 through SI-321 received 2 mgof anti-trypanosoma monoclonal antibody. Serum samples taken from eachanimal immediately prior to NVS3 injection were all negative foranti-sporozoite activity in IFAT and below the sensitivity of the(AGDR)₂ ELISA. Monkey IFAT ELISA Mean # Schizonts/ No. Titer (AGDR)₂μg/ml two 35 mm monolayers SI-74 6400 14.3 ± 8.9   54/1.5* SI-162(P)12,800   18.4 ± 14   44/0.5 SI-218 3200 4.3 ± 2.6 51.5/1   SI-250(P)6400 18.5 ± 5   49/0.5 SI-251(P) 6400 7.2 ± 3.6 37.5/1.5  SI-312(P) 64005.9 ± 3.5 36/0.0 SI-316 neg neg NT/31 SI-319 neg neg NT/10 SI-321 negneg NT/22 NT = not tested neg = below IFAT and ELISA sensitivity IFATtiters = reciprocal of dilutions (P) = protected in sporozoite challenge*sera before NVS3 injection/sera 1 hour after NVS3 injection

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

REFERENCES

1. Institute of Medicine. Malaria: Obstacles and Opportunities. S. C.Oaks, V. S. Mitchell, G. W. Pearson and C. Carpenter, eds. NationalAcademy Press, Washington D.C. (1991).

2. H. W. Mulligan, P. Russell and B. N. Mohan. J.Mal.Inst.India 4:25(1941).

3. R. S. Nussenzweig, J. Vanderberg, H. Most and C. Orton. Nature216:160 (1967).

4. D. F. Clyde, V. C. McCarthy, R. M. Miller and R. B. Hornick.Am.J.Med.Sci. 266: 398 (1973).

5. D. F. Clyde, V. C. McCarthy, R. M. Miller and W. E. Woodward.Am.J.Trop.Med.Hyg. 24:397 (1975).

6. D. F. Clyde, H. Most, V. C. McCarthy and J. P. Vanderberg.Am.J.Med.Sci. 266: 169 (1973).

7. K. H. Rieckmann et al. Trans.R.Soc.Trop.Med.Hyg. 68: 258 (1974).

8. K. H. Rieckmann, R. L Beaudoin, J. S. Cassells and D. W. Sell. Bull.W.H.O. 57: 261 (1979).

9. D. Herrington et al. Am.J. Trop.Med.Hyg. 45: 539 (1991).

10. J. E. Egan et al. Am.J.Trop.Med.Hyg. (1992) (In Press).

11. R. Edelman et al. J.Infect.Dis. 168: 1066 (1993).

What we claim is:
 1. A formulation protective against Plasmodium vivaxfor a time commensurate with the time monoclonal antibody Navy VivaxSporozoite 3 (HB10615) remains at pharmacologically active levels in asubject's blood stream, comprising a pharmaceutical amount sufficient toprovide passive immunization of Navy Vivax Sporozoite 3 (HB10615) in apharmaceutically suitable injectable solution.
 2. The formulation ofclaim 1 containing between about 2 and 30 mg of Navy Vivax Sporozoite 3(HB10615).
 3. The formulation of claim 1 containing about 2 mg of NavyVivax Sporozoite 3 (HB10615).
 4. A method of providing protection fromPlasmodium vivax induced malaria for subjects experiencing exposure toinfected mosquitoes, for a time commensurate with the time monoclonalantibody Navy Vivax Sporozoite 3 (HB10615) remains at pharmacologicallyactive levels in the subject's blood stream, that comprises introducingand circulating the antibody Navy Vivax Sporozoite 3 (HB10615) in thesubject's blood stream.
 5. The method of claim 4 wherein between about 2to 30 mg of Navy Vivax Sporozoite 3 (HB10615) is introduced into asubject's blood stream.
 6. The method of claim 4 wherein about 2 mg ofNavy Vivax Sporozoite 3 (HB10615) is introduced into a subject's bloodstream.
 7. The method of claim 4 wherein AGDR sites on the CS protein ofPlasmodium vivax are bound by the Navy Vivax Sporozoite 3 (HB10615). 8.A humanized antibody capable of providing passive protection againstPlasmodium vivax wherein said antibody has a variable region comprisingthe hyper variable regions of the heavy and light chains of monoclonalantibody Navy Sporozoite 3 (HB10615) and human antibody frameworkregions.
 9. A mouse/human chimeric antibody comprising the heavy andlight chain variable regions of monoclonal antibody Navy Sporozoite 3(HB10615) and a human constant region.